We plan to study the host-parasite relationship between Toxoplasma gondii and cultured mammalian cells. The approach will depend upon a combination of biochemistry and genetics. Using human fibroblast cells as the host, we shall isolate a series of temperature-sensitive mutants of T. gondii, particularly mutants that are defective in protein or nucleic acid synthesis. These mutants will be used to examine the lethal effect of T. gondii on its host cell and the role of the parasite in the function of its parasitophorous vacuole. We also plan to isolate and characterize a variety of drug-resistant mutants of T. gondii and use them in genetic crosses with the cat as the host. By using other unselected genetic markers we may be able to define linkage groups of the T. gondii genome. We shall attempt to establish the sexual cycle of T. gondii in cultured intestinal cells from kittens. This will allow the use of temperature sensitive mutants as genetic markers. By using host cell mutants with known biochemical defects we shall determine the importance of host metabolism to the parasite. We shall study host cells with defects in the synthesis of protein, cholesterol, unsaturated fatty acids, ATP, purines, pyrimidines, and amino acids. The synthesis of ribosomal and messenger RNA by intracellular T. gondii will also be studied in experiments not employing mutant host cells.